Description: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast, however few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A related processes in mitotic arrested cells. We identified 626 potential PP2ACdc55 substrates involved in a broad range of mitotic processes. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases’ consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases different than Cdk1. Indeed, Pkc1 and Cla4 kinases emerged as novel nodes of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in membrane trafficking and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome. In addition, we validated two new PP2ACdc55 substrates involved in early and late anaphase pathways, Slk19 and Lte1; and we also validated Zeo1, and other potential substrates, through protein interaction experiments. Finally, we performed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction.